Our research concerns the mechanism and consequences of Ty (Transposon yeast) element retrotransposition in the budding yeast Saccharomyces cerevisiae. Ty elements comprise five related families of long terminal repeat retrotransposons that transpose via an RNA intermediate. The Ty genome contains two genes, TYA and TYB, which correspond to the gag and pol genes of retroviruses, respectively. The retrotransposon is transcribed into a nearly genome-length RNA, which is the template for reverse transcription by the self-encoded reverse transcriptase protein and for translation. Ty protein maturation and reverse transcription take place within Ty virus-like particles (Ty-VLPs), which appear to be essential for the transposition process. Although Ty-VLPs accumulate in the cytoplasm, a sub-VLP preintegration complex containing Ty cDNA, the element-encoded integrase and perhaps other proteins probably is required to transit the nuclear membrane, and mediate integration at preferred chromosomal locations. We are particularly interested in the biology of Ty1 elements because these elements are the most abundant, competent for transposition, and their RNA transcripts accumulate to an exceptionally high level. Despite the abundance of Ty1 RNA, however, mature Ty1 proteins and VLPs are present at low levels, and Ty1 transposition events are also very rare. Although Ty1 elements preferentially integrate upstream of genes transcribed by RNA polymerase III, Ty1 insertions can mutate essentially any yeast gene, form large complex multimeric insertions of 100 kb or more, and can also initiate chromosomal deletions, inversions and translocations by homologous recombination with other Ty1 elements in the genome. Information gained from studying Ty elements has been successfully applied to several other areas of biomedical research. For example, understanding how Ty elements transpose in yeast has led to a greater understanding of how retroelements in other organisms including humans function, because many of these elements are related. Over 40% of the human genome is comprised of retroelement sequences, such as LINE and SINE, intracisternal A-type particle, and endogenous retroviral elements. Most importantly, genome rearrangements and insertional events involving these elements have been implicated in human disease and cancer. Further computational analyses of mammalian genome sequences coupled with functional genomic analyses of cancerous cells will likely reveal new roles for retroelements that can be modeled in yeast using Ty elements or their mammalian counterparts. In addition, many aspects of the retrotransposon replication cycle are similar to those of retroviruses, including HIV. Therefore, steps in the process of retrotransposition can be compared and contrasted with similar processes in retroviruses to learn more about both classes of elements. Over the past year, we have made progress in the following areas. We, in collaboration with an international consortium of yeast researchers headed by Dr. Mark Johnston (Washington University), have developed a near complete set (95% of all ORFs) of single gene deletions to systematically survey gene function. These mutations are currently being screened for their affects on Ty1 retrotransposition. In our continuing effort to identify cellular genes that modulate Ty1 retrotransposition, we have surveyed all members of the RAD2 family of nucleases, which are required for genome stability. We have shown that only Rad27/Fen1, a highly conserved structure-specific nuclease important for lagging strand DNA replication, inhibits Ty1 mobility by altering the fate of unincorporated cDNA. We have also discovered a novel form of post-transcriptional cosuppression that controls Ty1 element copy number. A decrease in the synthesis of Ty1 cDNA is strongly correlated with post-transciptional cosuppression. Unlike the conserved RNAi pathways that cosuppress transposable elements in a variety of eukaryotes, the level of Ty1 RNA increases with the addition of elements to the genome, yet genomic Ty1 retrotransposition decreases. In addition, Saccharomyces cerevisiae lacks the conserved RNAi pathway, therefore, identifying the factors responsible for Ty1 cosuppression may provide new insight into how eukaryotic genomes evolve and are stably maintained.